Patent EA-004428-B1 (EA)
Title: Modified exosomes and uses
Inventor: BENAROCH PHILIPPE
IPC: C07K14/705
Filed: 1999-11-04
Abstract: 1. Membrane vesicle produced by a genetically modified cell, wherein said vesicle comprising a recombinant molecule of major histocompatibility complex (CMH) of a human, comprising at least one alpha-chain of class II. 2. Membrane vesicle produced by a genetically modified cell, wherein said vesicle comprising a recombinant molecule of major histocompatibility complex of a human, comprising at least one beta-chain of class II. 3. Vesicle according to claim 1 or 2, characterized in that a recombinant molecule of major histocompatibility complex of class II comprises a alpha-chain and a beta-chain. 4. Vesicle according to any one of claims 1 to 3, characterized in that a recombinant molecule of major histocompatibility complex of class II is selected from serotypes DR-1-DR13, preferably DR1-DR7. 5. Vesicle according to any one of claims 1 to 4, characterized in that further comprises a molecule of major histocompatibility complex of class I. 6. Vesicle according to any one of claims 1 to 5, characterized in that comprises a complex between definite peptide and a recombinant molecule of major histocompatibility complex. 7. Vesicle according to any one of claims 1 to 6, characterized in that further comprises one or a plurality of interested heterologous molecules. 8. Vesicle according to any one of claims 1 to 7, characterized in that further comprises recombinant peptide or protein permitting its purification. 9. Vesicle according to any one of claims 1 to 8, characterized in that comprises a marker. 10. Vesicle according to claim 9, characterized in that the marker is green fluorescent protein (GFP). 11. Vesicle according to any one of claims 1 to 10, characterized in that it is essentially deprived of CMH endogene molecules. 12. Vesicle according to any one of claims 1 to 11, characterized in that comprises a recombinant molecule formed by fusing interested peptide and addressing signal. 13. Membrane vesicle, characterized in that it is obtained from genetically-modified mastocyte cell or derived genetically-modified mastocyte cell and comprises one or a plurality of interested heterologous molecules. 14. Vesicle according to claim 13, characterized in that the interested molecule is protein, polypeptide, peptide, nucleic acid, lipid or a substance of chemical, biological or synthetic origin. 15. Membrane vesicle according to claim 14, characterized in that a heterologous molecule is a molecule of major histocompatibility complex, an antigen, receptor ligand of receptor, receptor ligand, nucleic acid, a pharmaceutical product, a marker and/or peptide for purification. 16. Vesicle according to claim 15, characterized in that it expresses receptor ligand and comprises another interested heterologous molecule. 17. Membrane vesicle according to any one of claims 13 to 16, characterized in that comprises a recombinant molecule formed by fusing interested peptide and addressing signal. 18. A cell producing exosomes, characterized in that comprises one or a plurality of recombinant nucleic acids encoding a molecule of the major histocompatibility complex, comprising at least one (CMH) alpha-chain of class II of a human. 19. The cell according to claim 18, characterized in that it is a mastocyte cell. 20. The cell according to claim 19, characterized in that it descends from mastocyte cell line of basophile leucosis, in particular, from RBL, preferably RBL-2H3. 21. The cell according to any one of claims 18 to 20, characterized in that comprises recombinant nucleic acid encoding alpha-chain, or recombinant nucleic acid encoding alpha-chain and beta-chain of a molecule of the major histocompatibility complex of class II and, optionally, a molecule of the major histocompatibility complex of class I. 22. A process for producing a membrane vesicle according to any one of claims 1 to 17, comprising a definite recombinant molecule, comprising at least one (CMH) alpha-chain of class II of a human including the following steps: a) culturing a mastocyte cell or a derived mastocyte cell, comprising recombinant nucleic acid encoding said definite recombinant molecule; c) recuperation of vesicles produced by said cells, said vesicles comprise said definite recombinant molecule. 23. The process according to claim 22, characterized in that comprises an intermediate step b) in the course of which cells are stimulated for induction and/or increasing exosome secretion. 24. The process according to claim 22 or 23, characterized in that a definite recombinant molecule is exposed exterior of exosome or included, partially or completely, in exosome cytosolic fraction. 25. The process according to any one of claims 22 to 24, characterized in that a recombinant molecule is a molecule of the major histocompatibility complex, an antigen molecule, receptor ligand of receptor, receptor ligand, peptide for purification or other interested polypeptide. 26. The process according to any one of claims 22 to 24, characterized in that nucleic acid comprises, besides, a region encoding addressing signal to membrane compartments of a mastocyte cell. 27. A process for producing exosome, comprising a complex peptide-CMH of a definite composition, comprising: - culturing a producing exosome cell, comprising one or a plurality recombinant nucleic acids, encoding a definite CMH recombinant molecule, comprising at least one (CMH) alpha-chain of class II of a human; - stimulation of cells for inducing of exosome liberation; - recuperation of exosomes produced by said cells, said exosomes express on its surface said definite recombinant CMH molecule; and - contacting exosomes with peptide or peptides. 28. The process according to claim 27, characterized in that a producing cell is a mastocyte cell or derived mastocyte cell. 29. The process according to any one of claims 27 to 28, characterized in that the producing cell is essentially deprived of CMH endogene molecules. 30. A process for producing exosome, comprising a complex peptide-CMH of a definite composition, comprising: - culturing a producing exosome cell, comprising one or a plurality recombinant nucleic acids, encoding a definite CMH recombinant molecule, and nucleic acid, including a region encoding definite recombinant peptide; - stimulation of cells for inducing of exosome liberation; - recuperation of exosomes produced by said cells, said exosomes express on its surface said definite recombinant CMH molecule, associated with said recombinant peptide. 31. The process according to claim 30, characterized in that nucleic acid encoding recombinant peptide encodes a derivative of an invariant li chain, in which CLIP region is deleted and substituted by said peptide. 32. The process according to any one of claims 30 to 31, characterized in that a producing cell is a mastocyte cell or derived mastocyte cell. 33. The process according to any one of claims 30 to 32, characterized in that the producing cell is essentially deprived of CMH endogene molecules. 34. A process of modification of exosome composition, comprising: - introduction in a cell producing exosome nucleic acid encoding a definite molecule associated with addressing signalin membrane compartments; and - production of exosomes from said cell. 35. A composition, comprising one or a plurality of membrane vesicles according to any one of claims to 17. 36. Use of a vesicle according to any one of claims to 17 for production polyclonal and/or monoclonal antibodies. 37. A process for producing antibodies, comprising immunization of an animal with vesicle according to claim 7 and recuperation of antibodies and/or cells, producing antibodies or taking part in immune response. 38. A process according to claim 37, characterized in that it is used for producing monoclonal antibodies, in particular specific to CMH-peptide combination. 39. Use of an antibody or a fragment thereof, obtained according to claim 37 or 38 for detection the presence of corresponding specific antigens in a biological sample. 40. Use of an antibody or a fragment thereof, obtained according to claim 37 or 38 for the preparation of a therapeutic composition for inhibiting interaction between T-lymphocyte receptor and complex CMH-peptide to which is specific. 41. Use of vesicle according to claim 1 or 2 for the preparation of a therapeutic composition for inhibiting interaction between T-lymphocyte receptor and complex CMH-peptide to which is specific. 42. Use of membrane vesicle according to any one of claims 1 to 17 for detection in vitro or ex vivo in a biological sample of partners, specific to a protein molecule. 43. Use according to claim 42, characterized in that exosome carries a complex CMH-peptide for detection in a biological sample T-lymphocytes specific to said complex. 44. Use according to claim 42, characterized in that exosome carries a TcR receptor for detection in a biological sample T-lymphocytes specific to said complex. 45. Use according to claim 42, characterized in that exosome carries a receptor of ligand for detection in a biological sample the presence of said ligand. 46. A method of detection in vitro or ex vivo the presence of T-lymphocytes specific to a complex antigen-CMH in a biological sample, comprising contacting said sample with a marked exosome according to claim 7, comprising said complex antigen-CMH and detecting of T-lymphocyte marking in said sample. 47. Use of vesicle according to claim 6 for clonal amplification and/or stimulation ex vivo cytotoxic or regular T-lymphocytes. 48. Use of a vesicle according to any one of claims 13 to 17 for the preparation of a composition intended for delivering said molecule to a cell. 49. A composition, comprising one or a plurality of vesicles according to any one of claims 1 to 17 immobilized on a support. 50. The composition according to claim 49, comprising one or a plurality of membrane vesicles, are immobilized on a ball, in particular, on a latex ball or on a magnetic ball. 51. Use of a membrane vesi